The objective of this work is to investigate the role of the HIV-1 nucleocapsid (NC) and reverse transcriptase (RT) proteins, as well as human APOBEC3 viral restriction factors, in the regulation of reverse transcription in retroviral systems The proposed work combines single molecule methods with biochemical methods and measurements in cells to obtain a complete understanding of nucleic acid (NA) interactions involved in retroviral replication. We use these methods to probe the mechanisms by which retroviral proteins dynamically restructure and organize NA to facilitate replication, and to determine how these processes are regulated. To do this, the PI has pioneered single molecule NA stretching methods that quantitatively probe NA structural rearrangements and protein-NA interactions. In the previous cycle, we demonstrated that the capability of NCs to rearrange NAs, referred to as NA chaperone activity, is directly correlated with HIV-1 replication in cells. The proposed work seeks to understand how this chaperone activity targets specific structures and facilitates NA reorganization without interfering with reverse transcription. In contrast to NC's facilitation of reverse transcription, human APOBEC3 (A3) proteins may inhibit reverse transcriptase activity. We will probe the DNA interactions of several A3 proteins and directly monitor RT activity with A3 proteins and NC. The specific aims are: (1) To determine how NC alters the conformational landscape of specific DNA and RNA structures. To test the hypothesis that HIV-1 NC is optimized to function as a chaperone with specific NA structures, we will directly measure the folding landscape of specific RNA and DNA structures in the absence and presence of wild type and mutant NC, determining the components of RNA and protein structure that optimize chaperone activity by lowering the unfolding barrier. (2) To probe the mechanism of APOBEC3 protein binding to single-stranded DNA. We hypothesize that different A3 proteins inhibit retroviral and retrotransposon replication both by acting as active enzymes that deaminate genomic sequences, as well as in a deaminase-independent manner. To test this hypothesis, we will probe the ssDNA binding activities of three representative A3 proteins: the primarily monomeric A3A, as well as wild type and mutant A3G and A3F, which both oligomerize. We will compare the results with HIV-1 replication inhibition by the same proteins in cells. (3) To mechanically measure the effects of HIV-1 NC and human APOBEC3 proteins on single DNA molecule elongation by HIV-1 reverse transcriptase. We will directly measure the elongation of single DNA molecules by RT in the absence and presence of wild type and mutant NC as well as A3 proteins with different oligomerization properties to determine the extent to which these protein enhance or inhibit RT activity.